Multiple antibiotic resistance in bacteria has become a clinical albatross. One of the mechanisms by which the enteric bacteria can become resistant to a wide spectrum of antibiotics is through induction of the mar (multiple antibiotic resistance) locus which specifies an autorepressor, MarR, and a transcriptional activator, MarA. Precisely how transcriptional activators work is one of the major theoretical problems of both prokaryotic and eukaryotic gene regulation. MarA is a member of the large AraC family of transcriptional activators. Though known for over 30 years, none has been structurally defined by x-ray crystallography or NMR. In collaboration with J. L. Rosner, S. Rhee and D. Davies, we have obtained co-crystals of MarA with a DNA oligonucleotide of the mar promoter which defract to 2.9 A. We also have prepared isomorphous crystals substituted with iodine and selenium which are currently being analyzed. In studying the transcriptional activation of the mar promoter by MarA in collaboration with J. L. Rosner we found that the small DNA binding protein Fis acts as an accessory factor. That is, Fis does not stimulate the promoter unless MarA (or the closely related activators SoxS or Rob) is also present, although the activators partially stimulate the promoter even in the absence of Fis. In addition, we have obtained mutants of MarA that stimulate mar transcription but cannot be further stimulated by Fis. We have postulated that these mutants of MarA might bind to the promoter but not bend it. We are currently testing this hypothesis. Since the mar promoter is a Class I promoter (i.e. it binds MarA at a site upstream of the RNA polymerase binding site) its activation by MarA might differ from that of other promoters that bind MarA at the RNA polymerase site (Class 2 promoters). (CRP, a different activator, which unlike MarA stimulates as a dimer rather than a monomer, has different sites of interaction with RNA polymerase when acting on Class I and Class II promoters.) We have therefore been studying two Class 2 promoters activated by MarA, inaA and micF. The limits of these promoters have been defined by deletion mapping and in vitro transcription studies have been initiated. One of the problems of NMR imaging of DNA binding proteins is obtaining C13 and N15 derivatized oligonucleotides. In collaboration with J. Louis, A. M. Gronenborn and G. M. Clore, I have developed a PCR technique for obtaining substituted oligonucleotides in high yield and of sufficient purity for NMR studies.